The ubiquitin-dependent proteolytic process regulates many short lived intracellular proteins, whose concentrations change promptly as the result of alterations in cellular physiological conditions. See Hochstrasser, M. et al. (1996) Annu. Rev. Genet. 30, 405-439.; King, R. W., et al. (1996) Science 274, 1652-1659; Hershko, A. et al. (1997) Curr. Opin. Cell Blot. 9, 788-799. In addition to performing “housekeeping” functions such as homeostasis and the removal of misfolded proteins, this proteolytic process is involved in the degradation of many regulatory proteins, such as cyclins, CDK inhibitors, transcription factors, and signal transducers. In brief, ubiquitin-mediated proteolysis begins with activation of ubiquitin, a 76-amino acid protein expressed in all eukaryotic cells, in an ATP-dependent manner by an ubiquitin-activating enzyme (E1 or Uba). The activated ubiquitin forms a high energy thiolester bond with E1 and is passed to a cysteine residue also via a thiolester bond within an ubiquitin-conjugating enzyme designated as an E2 or Ubc. E2-linked ubiquitin is then transferred to a side chain amino group of a lysine residue in the substrate to form a terminal isopeptide bond, either directly or often indirectly targeted by a ubiquitin ligase known as E3. Substrate proteins can be linked to a single ubiquitin (monoubiquitinated) or multiple ubiquitin molecules (polyu biquitinated). The significance of monoubiquitinated conjugates is not clear since they do not appear to be short-lived. Successive covalent ligations of additional ubiquitins to the Lys 48 of the preceding ubiquitin via an isopeptide bond results in polyubiquitinated conjugates which are rapidly detected and degraded by the 26S proteosome. E3 is functionally, rather than structurally, defined as an ubiquitin ligase activity that is both necessary and sufficient for transfer of ubiquitin from a ubiquitin-charged E2 to a substrate, and is further believed to be involved in many polyubiquitination reactions by providing substrate specificity. Because most polyubiquitinated proteins are indiscriminately delivered to the 26S proteosome for degradation, elucidating the mechanism and regulation of E3 ligase activities has become a critical issue central to the understanding of regulated proteolysis.
The cullin family of protiens potentially form a large number of distint E3s as indicated by the existence of a multi-gene family and by the assembly of yeast CDC53 into at least three distinct E3 complexes: with SKP1-CDC4, with SKP1-GRR1 and likely with SKP1-MET30 to mediate the ubiquitination of SIC1, CLN and SWE1 proteins, respectively. See, e.g., Skowyra, D., et al.,(1997) Cell 91, 209-219; Feldman, R. M. R. (1997) Cell 91,221-230; and Kaiser, P. et.al., (1998) Genes & Dev. 12, 2587-2597. Through targeting different substrates, different cullins function in a variety of diverse cellular processes. For example, CDC53 is required for S phase entry (Mathias, N. et al., (1996) Mol. Cell Biol. 16, 6634-6643; for coupling glucose sensing to gene expression and the cell cycle (Li, F. N. and Johnston, M. (1997) EMBO J. 16, 5629-5638; and possibly for activating mitotic CLB-CDC28 activity (Kaiser, P.et al., (1998) Genes & Dev. 12, 2587-2597). As set forth in more detail below, the C. elegans cul-1 mutant displays a hyperplasia phenotype. Human CUL2 is associated with the tumor suppressor VHL (von Hippel-Lindau) implicated in the regulation of the stability of hypoxia-induced mRNA (see Pause, A., et al., (1997) Proc. Natl. Acad. Sci. USA. 94, 2146-2161; Lonergan, K. M. et al., (1998) Mol. Cell Biol. 18, 732-741. Human CUL4A is implicated in oncogenesis by its genomic amplification and overexpression in breast cancers (Chen, L-C., et al., (1998) Cancer Res. 58, 3677-3683), and deficiency of the cullin-related APC2 results in mitotic arrest (Zachariae, W. et al., (1998) Science 279, 1216-1219; Yu, H., et al., Current Biology 6, 455-466).
The knowledge of E3 ubiquitin ligases is presently limited. Among the few characterized E3 ligases are the N-end rule ubiquitin ligase E3α/Ubr1 that recognize proteins by binding to the basic or hydrophobic residues at the amino-termini of substrate proteins (reviewed in Varshavsky, A. (1996) Proc. Natl Acad. Sci U.S.A. 93, 12142-12149); the HECT (homologous to E6-AP carboxy terminus) domain proteins represented by the mammalian E6AP-E6 complex which functions as a ubiquitin-ligase for p53 (see Scheffner, M. et al., (1993) Cell 75, 495-505; Huibregtse, J. M., et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2563-2567; Scheffner, M.et al. (1995) Nature 373, 81-83); and the APC (anaphase-promoting complex or cyclosome), a 20S complex that consists of 8 to 12 subunits and is required for both entry into anaphase as well as exit from mitosis (see King, R. W., Deshaies, Science 274, 1652-1659).
The APC plays a crucial role in regulating the passage of cells through anaphase by promoting ubiquitin-dependent proteolysis of many proteins. The APC destroys the mitotic B-type cyclin for inactivation of CDC2 kinase activity and initiating cytokinesis. The APC is also required for degradation of other proteins for sister chromatid separation and spindle disassembly, including the anaphase inhibitors PDS1 (Cohen-Fix, O., et al.(1996) Genes & Dev. 10, 3081-3093) and CUT2 (Funabiki, H., et al. (1996) Nature 381, 438-441), ASE1 (Juang, Y-L. et al. (1997) Science 275, 1311-1314) and the cohesion protein SCC1P (Michaelis, C. et al., (1997) Cell 91, 35-45). All known proteins degraded by the APC contain a conserved nine amino acid stretch commonly known as the destruction box that is necessary for their ubiquitination and subsequent degradation (Glotzer, M., et al. (1991) Nature 349, 132-138). Proteins that are degraded during G1, ranging from G1 cyclins and CDK inhibitors to transcription factors, do not contain the conserved destruction box or any other common structural motif. Instead, substrate phosphorylation appears to play an important role in targeting their interaction with E3 for subsequent ubiquitination. Genetic and biochemical analysis has identified in yeast an E3-like activity, dubbed as the SCF, that plays a key role in regulating G1 progression. The SCF consists of at least three subunits, SKP1, CDC53/cullin and an F-box containing protein, in which SKP1 functions as an adaptor to connect CDC53 to the F-box protein which binds directly to the substrate (Feldman, R. M. R., et al., (1997) Cell 91, 221-230; Bai, C., et al. (1996) Cell 86,263-274; Willems, A. R., (1996) Cell 86, 453-463; Verma, R.(1997) Science 278, 455-460; Skowyra, D., (1997) Cell 91, 209-219).
In a screen for mutants with excess postembryonic cell divisions in C. elegans, the gene cullin-1 (CUL1), was identified. Loss of function of this gene caused hyperplasia of all tissues as a result of the failure to properly exit from the cell cycle. See Kipreos, E. T., et al., (1996) Cell 85, 829-839. CUL1 represents an evolutionarily conserved multigene family that includes at least seven members in C. elegans, six in humans, and three in budding yeast including Cdc53p (Kipreos, et al., supra, and Mathias, N. et al., (1996) Mol. Cell Biol. 16, 6634-6643). Like yeast CDC53, human cullin 1 directly binds to SKP1 to form a multi-subunit complex with SKP2 (an F box protein), cyclin A and CDK2 (Lisztwan, J. et al., (1998) EMBO J. 17,368-383; Michel, J. and Xiong, Y. (1998) Cell Growth. Differ. 9, 439-445; Lyapina, S. A., et al. (1998) Proc. Natl. Acad. Sci. USA 95, 7451-7456; and Yu, Z. K. et al. (1998) Proc. Natl. Acad. Sci U.S.A. 95, 11324-11329), and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Recently, a subunit of the mitotic APC E3 complex, APC2, was found to contain limited sequence similarity to CDC53/cullins (Zachariae, W. et al., (1998) Science 279, 1216-1219; Yu, H. et al., (1998) Science 279, 1219-1222.). These findings, together with the fact that no obvious structural similarity between other components of the SCF and APC complexes exists, underscore an important and conserved role for cullin proteins in ubiquitin-mediated proteolysis, possibly as an intrinsic partner of ubiquitin ligases. However, despite extensive investigations of the APC and SCF E3 ligases, the nature of ubiquitin ligases has thus far been elusive. It still remains to be determined whether there is a “ligase” in the APC and SCF. Whether the cullin proteins act as ubiquitin ligases to catalyze isopeptide bond formation or as scaffold proteins to bring together E2-Ub and substrates together is heretofore not described.
Equally important as the mechanism that determines the substrate specificity is the regulation of E3 ligases, which is presently poorly understood. The activity of the APC is cell-cycle regulated, and active from anaphase until late G1. See Amon, A. (1994) Cell 77, 1037-1050; King, R., et al., (1995) supra; Brandeis, M. and Hunt, T. (1996) EMBO J. 15, 5280-5289. The principle regulation is probably provided by subunit rearrangements such as CDC20 and CDH1 binding (Visintin, et al., (1997) Science 278, 460-463; Schwab, M. (1997) Cell 90, 683-693; Sigrist, S. J. and Lehner, C. F. (1997) Cell 90, 671-681; and Fang, G. (1998) Mol. Cell 2, 163-171). Phosphorylation of certain subunits may also play an important, but supplementary role (Lahav-Baratz, S., Proc. Natl. Acad. Sci. USA 92, 9303-9307; Peters, J.-M. et al. (1996) Science 274, 1199-1201). Regulation of CDC53 and cullin-mediated E3 ligase activity during interphase is heretofore not described.